Acinetobacter baumannii has become a serious nosocomial pathogen due to its persistence in the hospital environment and its multi-drug resistance patterns. Outbreaks of multidrug resistant A. baumannii (MDRAB) have been reported in many hospitals worldwide. What is surprising about these reports is many outbreaks are polyclonal; that is, multiple phenotyically unique strains may appear in an outbreak. This is in contrast with outbreaks caused by other bacteria that are typically monoclonal caused by dissemination of a single strain of bacteria. From May to December 2007 a polyclonal outbreak of multiple multidrug-resistant Acinetobacter baumannii (MDRAB) occurred at the NIH Clinical Center. In 2008 and 2009 similar outbreaks were observed primarily in ICU patients, typically beginning in the warm summer months and persisting for 4-6 months. Analysis of the strains from each seasonal outbreak by conventional strain typing techniques demonstrated what appeared to be multiple strains of MDRAB circulating in the ICU populations. A series of experiments were performed to determine if the outbreak strains were polyclonal or originated from a common ancestor strain and then underwent genome rearrangement. A total of 45 A. baumannii clinical and surveillance isolates from the 2007 outbreak, including 29 MDRAB, were studied. Epidemiological relatedness of these strains was analyzed using rep-PCR, pulse field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). A total of 17 different pulsotypes were identified by PFGE, but only 4 were associated with MDRAB. Among these, one pulsotype presented a distinct temporal trend, displacing all other pulsotypes at the end of the outbreak. Rep-PCR was not able to differentiate most of the MDRAB. On the other hand MLST, showed a close relationship between three of MDRAB pulsotypes. This result is consistent with a common ancestor for the epidemic MDRAB strains in this outbreak. This increased genome plasticity within epidemic MDRAB might be associated with insertion elements. This hypothesis will be tested by whole genome sequencing. Five strains were selected representing the 3 most common pulsotypes in the 2007 outbreak and 2 strains that developed colistin resistance. Whole genome sequencing for each of the 5 strains was performed and assembly and analysis of the genomes is currently underway. Although other Acinetobacter genomes have been sequenced, this is the first example where strains responsible for an well-defined outbreak have been compared. We are confident that the results of this effort will determine if Acinetobacter is unique in its ability to undergo rapid genome rearrangements leading to increased virulence and the ability to acquire genes encoding antibiotic resistance.